Nanolipidic particles

ABSTRACT

Nanolipidic Particles (NLPs) having average mean diameters of 1 nm to 20 nm are made from a precursor solution. NLPs can be loaded with a desired passenger molecule. Assemblies of these particles, called NLP assemblies, result in a vehicle population of a desired size. Single application or multifunction NLP assemblies are made from the loaded NLPs and range in size from about 30 to about 200 nm. A method of using preloaded NLPs to make larger carrier vehicles or a mixed population provides increased encapsulation efficiency. NLPs have application in the cosmetics, pharmaceutical, and food and beverage industries.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/755,171 filed Dec. 30, 2005.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

TECHNICAL FIELD OF INVENTION

This invention concerns the field of incorporating passenger moleculesin lipid vesicles.

BACKGROUND OF THE INVENTION

Incorporating passenger molecules, such as pharmaceutical activeingredients, in lipid vesicles such as liposomes has been reported inthe prior art. An amphipathic carrier structure denoted as a SolventDilution Microcarrier (“SDMC”) was disclosed in U.S. Pat. No. 5,269,979.In general, the '979 patent described making a plurality of SDMCvehicles by solubilizing an amphipathic material and a passengermolecule in a first quantity of a non-aqueous solvent. Following this, afirst quantity of water was added, forming a turbid suspension. In asubsequent step, a second quantity of non-aqueous solvent was added toform an optically clear solution. The final step of a preferredembodiment was to organize the optically clear solution into SDMCvehicles by mixing with air or a second quantity of water.

In U.S. Pat. No. 5,879,703, a method for preparing a shelf-stableprecursor solution useful for remote encapsulation of active ingredientswas described. In '703, the precursor solution was made by solubilizingan amphipathic material in a non-aqueous solvent. A quantity of waterwas added to the first mixture to form a precursor solutioncharacterized by optical clarity and being monophasic at roomtemperature. The precursor solution could be stored for an extendedperiod of time—and the desired active ingredient added at a later time,perhaps at a remote location, to form a loaded precursor solution. SDMCscould be formed, in preferred embodiments, from the loaded precursorsolution by diluting with water or mixing with air. SDMCs ranged fromabout 230 to about 412 nanometers in size.

Although SDMCs and the shelf-stable precursor solution provided formaking vehicles suitable for delivering active ingredients in a varietyof applications, a need remained for improved vehicles for delivery ofpassenger molecules.

It has now been found that the shelf-stable precursor solution such asdescribed in the '703 patent can be used as a starting material in anovel method which results in vehicles of a smaller size than previouslyreported. The starting material is manipulated by dilution with anon-aqueous solvent, either before or after loading with a passengermolecule, to provide one or more defined populations of nanolipidicparticles (“NLPs”) which range in size from about 1 nanometer to about20 nanometers. NLP assemblies are formed from the NLPs which range insize from about 30 nanometers to about 200 nanometers. In addition, ithas been found that NLPs can be used in a method for making carriervehicle preparations which are mixed smaller and larger carriervehicles, or having a larger mean size of about 200-300 nanometers, butimproved encapsulation of passenger molecules.

DETAILED DESCRIPTION

Nanolipidic particles (“NLP”s) according to the present invention have asize range of from about 1 to about 20 nanometers, as measured by astandard laser light scattering technique, discussed in detail herein.Various subpopulations of NLPs may be made. The preferred distinctsubpopulations of NLPs range in size from about 1 to about 4 nm, fromabout 4 to about 7 nm, from about 7 to about 10 nm, from about 10 toabout 14 nm, from about 14 to about 18 nm, and from about 18 to about 20nm. A preferred subpopulation comprises NLPs having an average size ofabout 9 to 10 nanometers.

NLPs are made from a precursor solution as described in U.S. Pat. No.5,879,703, which is herein incorporated by reference as if fully setforth herein. As stated in the '703 patent, a precursor solution may bemade by solubilizing an amphipathic material in a first quantity of anon-aqueous solvent appropriate to solubilize the amphipathic materialto form a first mixture. The amphipathic material preferably comprisesphospholipids (PL). A preferred PL comprises one or more of thefollowing phosphatides: phospatidylcholine (PC), phospatidylethanolamine(PE), phosphatidic acid (PA) and phosphatidylinositol (PI). In apreferred embodiment, PC, PE, PA and PI are combined. A preferred ratioof PLs useful in the invention is PC:PE:PA:PI of 6.5:2.5:0.7:0.3 inethanol. Preferably, one gram of PL is solubilized in 5.0-7.5 ml ofethanol solvent.

After dissolution of the amphipathic material, a quantity of water isadded to form a turbid suspension. The amount of water to add isapproximately 9 kg to 31 kg of dissolved amphipathic material, but canbe varied to result in the desired turbid suspension. A second quantityof non-aqueous solvent, such as ethanol, is added until the turbidsuspension is monophasic and has optical clarity at room temperature.This resulting product is a precursor solution which is shelf-stableover time.

In the '703 patent, it was disclosed that a precursor solution madeaccording to the process disclosed therein was shelf stable at least upto two years, and perhaps longer, as long as it remains in a monophasiccondition. It has been recently determined that precursor solutions madeby this method are stable for at least eight years, independent ofmanufacturing, location, season, year and lot.

It has now been found that a precursor solution such as disclosed in'703 can be used as a starting material to make nanolipidic particles(NLPs) and NLP assemblies. In '703, the precursor solution was disclosedas being useful for making SDMCs at a later point in time and, perhaps,a remote location. SDMCs have a diameter of from about 230 to about 412nm. In contrast, NLPs have a mean diameter of from about 1 nm to about20 nm and NLP assemblies have a mean diameter from about 30 nm to about200 nm.

Various populations of NLP assemblies may be made for variousapplications. Preferred populations range from about 40-60 nm; about60-80 nm; about 80-110 nm; about 110-140 nm; and about 150-200 nm. NLPassembly populations are generally 20-30% smaller in diameter than SDMCsfor the same passenger molecule.

A slightly larger population or mixed population of carrier vehicles isreferred to herein as ECVs or encapsulating carrier vehicles. Althoughoverlapping the mean diameter of SDMCs, the ECV is made using adifferent method employing NLPs and the result is a carrier vehiclepopulation which has been found to exhibit a higher encapsulatingefficiency. The ECVs are described as having a mean diameter from about200 nm to 300 nm.

To make carriers for passenger molecules according to the disclosedmethod, such as NLP populations, NLP assemblies, or ECVs, the precursorsolution as previously described in the '703 patent is diluted with asuitable solvent or mixed solvent system which is compatible with thesolvent system used in the precursor solution. This dilution isperformed either before or after addition of the passenger molecule aswill be further described in detail below. The solvent is selected forbiocompatibility if the end use of the carriers will require thatcharacteristic. The solvent or mixed solvent system used for dilutionmust be miscible with the solvents in the precursor solution and shouldbe effective to disperse rather than dissolve the carriers. Mostpreferably, the solvent used for dilution is ethanol, since it possessesthe desired qualities. The dilution is preferably conducted in asequential or serial manner. For example, a first dilution of 1:10provides a population of carriers, and further serial dilution to about1:0.5 provides a series of populations of carriers. The size of thecarriers in each dilution can be determined by laser light scattering.Mixed populations of NLPs and larger vesicles may be created at lowerdilutions with the non-aqueous solvent. An appropriate instrument forthis purpose is the Zetasizer 1000 manufactured by Malvern Instruments,(Worcestershire United Kingdom). Diameters of particles reported hereinwere determining using the Multimodal Analysis Mode of the Zetasizer1000 to determine particle size by peak intensities. Other techniquesmay be used to analyze particle size, which results can be correlated tothe numerical values obtained with the light scattering techniquedescribed herein.

Addition of the desired passenger molecule occurs prior to dilution withthe solvent if the passenger molecule is lipophilic or amphipathic.Addition occurs after dilution of the passenger molecule is watersoluble.

Thus, in the case of a lipophilic or amphipathic passenger molecule, theNLP loaded populations form upon dilution with the solvent. NLP assemblypopulations or ECVs are formed by dilution of the NLP loaded populationinto water.

In the case of a water soluble passenger molecule, the precursorsolution is mixed with a passenger molecule dissolved in water. NLPassembly populations or ECVs are formed upon dilution with thenon-aqueous solvent. If a serial dilution technique is used, distinctpopulations are formed.

Based on curves observed from different classes of compounds, ranges forthe finished NLP assembly population can be established for each NLPpopulation used to form the final NLP assembly population. The morenon-aqueous solvent that is used to dilute the NLPs, the smaller the NLPassembly populations.

Various NLP loaded populations may be mixed and matched to provide amultifunctional NLP assembly product. As just one example, in a skincare product, it might be desired to topically provide Vitamin E and anantibiotic in a single preparation. The different NLP loaded populationswithin the NLP assembly could provide a preparation which allows oneactive ingredient to be preferentially absorbed over the other, thusallowing a control of the rates of penetration of different ingredientsin a single preparation. Alternatively, a single NLP population could beloaded with more than one passenger molecule to provide themultifunctionality.

An injectable multifunctional NLP assembly product could also provideadvantages: Due to the novel ability of this invention to produce welldefined populations of extremely small (sub 150 nm) populations, NLPproducts may be able to be produced which have greatly increased serumhalf-lives. Such particles may be able to evade initial uptake by thebody's immune system and possibly aid in the delivery of drug payloadsto cells and tissues not easily accessed by normal vascular deliverymechanisms; i.e. trans blood-brain barrier delivery and extra-vasculardelivery of drug payloads to selected or targeted organs and tissues.

The ability to select an NLP population of a preferable size for a givenapplication provides advantages to the manufacturing process as well.Less material will be required to form the end product if an NLPprecursor solution is selected for a particular size need. Loadingefficiency also goes up. The number of NLP particles increases as thesize of the NLP populations decreases as a function of the decreasingdiameter of the spherical NLP product (assuming the amount of lipid topassenger molecule remains constant). This may provide a higherconcentration of passenger molecule per unit volume.

Another advantage to the NLP technology is that an optically clearsolution containing NLPs loaded with passenger molecules can be made byselecting conditions where the NLPs are less than about 150 nm in size.It is many times important that a product appear optically clear or itwill fail to gain consumer acceptance. For example, loaded NLPs in anoptically clear solution have application in the beverage industry andthe pharmaceutical industry for liquid products. As one example, amouthwash can be prepared that contains NLPs which encapsulates aningredient for time-release in the mouth. A consumer prefers to purchasean optically clear mouthwash rather than a cloudy one.

The passenger molecules suitable for use in forming a NLP loadedpopulation are numerous. In one embodiment, passenger molecules can beselected which exhibit lipid solubility or are amphipathic. Thesemolecules have solubility profiles ideally suited for loading into NLPS.In another embodiment, water soluble molecules may be incorporated intoNLPs by solubilization into the aqueous solution used to form thefinished NLP product. Using these two approaches virtually any moleculemay be incorporated as a passenger molecule into NLP products of definedsizes. An innovative use of both approaches may be used to incorporateboth lipid and water soluble compounds into a NLP assembly product byfirst incorporating lipid soluble compounds into NLPs prior to dilutionwith ethanol and second incorporating water soluble molecule(s) into thewater solution used to form the finished NLP product of defined size.

Numerous passenger molecules have been incorporated into NLPs. Forexample, fat-soluble vitamins may be used as a passenger molecule.Vitamins D, E and K have been found to be appropriate for NLPs.

Water soluble vitamins, such as Vitamin B and C may also be used aspassenger molecules. Both water soluble and fat soluble vitamins may becombined in an NLP assembly if it is desired that both be administeredby using the technique discussed above.

Passenger molecules such as are useful in sunscreens may also be used.Benzophenone (UVB and UVA absorber), for example, can be appropriatelyused as an NLP passenger molecule.

Other preferred passenger molecules are antibiotics such asaminoglycosides (Gentamycin), beta-lactams (Penicillin G) and macrolides(Erythromycin). Anesthetics such as lidocaine have been effectivelyincorporated in NLPs, as have steroids and antifungals (griseofulvin).

NLPs may also be used in the food and beverage industry. For example,NLPs incorporating caffeine may be used in dietary supplements forappetite suppression. Encapsulation in NLPs has been found to beeffective to mask the taste of the passenger molecule if it is desiredthat tasting of such be bypassed upon ingestion.

Another application in the food and beverage industry is theincorporation of substances into NLPs which will be tasted, rather thanmasked. Flavorings such as peppermint oil and other oils areappropriately incorporated into NLPs The encapsulation of oil-containingsubstances may lead to increased shelf life in that the encapsulatedsubstance is protected from oxidation. In addition, the encapsulation ofsubstances would permit additional options for manufacturers andconsumers. As just one example, a manufacturer of a beverage couldprepare and bottle one base flavor. The consumer would then have theoption of adding NLP packets to the beverage to meet the tastepreferences of the consumer or to enrich it with vitamins. A consumerthat prefers a strong peppermint flavoring in a chocolate drink couldadd NLPs containing peppermint oil to his or her beverage. Substancesthat are meant to be tasted can also be loosely associated with theexterior of the NLP by providing such substances in the aqueous phase ofthe procedure. For example, an NLP containing a vitamin that preferablyshould not be tasted can have a pleasant taste on the outside thereof.If it is desired that the NLPs remain in the mouth so that theircontents can be tasted, a natural carbohydrate or sugar can be linked tothe NLP by merely providing it in the aqueous solution. This will stickto the inside of the mouth for a period of time, and normal mouthchemistry and mastication will release the contents of the NLPs toprovide the desired effect. The NLPs can also be subjected to agitationand shear such as in a blender or heavy industrial equipment at amanufacturing site to provide flavorings to foods and beverages.

NLPs may also be used for incorporation of peptides. Tripeptides,Tetrapeptides, Hexapeptides and Nonapeptides have been effectivelyincorporated into NLPs.

NLPs may also be useful in industries requiring dye as an ingredient.Water soluble and lipid soluble dyes may be used.

NLPs may be used to incorporate oils of various types, such as essentialoils and scented oils, into products. Examples of such products arelotions, emulsions, creams, cosmetics, cosmeceuticals and perfumes. TheNLPs may be loaded with the oils and provide a time-release function tothe product, allowing the desired ingredient to be released over aperiod of time. For example, the scent of a perfume could beincorporated in one or more size populations of NLPs and the scentreleased over a longer period of time than scent not incorporated inNLPs.

If the desired passenger molecule is water soluble, the passengermolecule should first be dissolved in water. The incorporation step, orloading of the passenger molecule into the NLP, is accomplished when theNLP product is formed by adding the dissolved passenger molecule to theprecursor solution.

Example 1 Use of Shelf-Stable Precursor Solution as a StartingIngredient for Nanolipidic Particles

Shelf-Stable Precursor Solution (Manufacturing Lot 0013-040010) was usedto prepare NLPs of a variety of defined size populations. Using theMalvern Laser Light Scattering instrument (Zetasizer 1000) nascentpopulations of NLP were measured to be 9 nm. To prepare NLP assemblypopulations of defined sizes, the following procedure was followed: 1 mlaliquots of Stock Precursor Solution (Lot 0013-040010) were placed in 10ml test tubes. To prepare defined populations of NLP product variousamounts of ethanol were added to each of the sample preparations.Following formation of the NLP preparations, 1 ml aliquots of eachsample were added to 20 ml of distilled water using a 2 ml pipette. Eachpreparation was stirred at room temperature for 5 minutes and then 2 mlaliquots were removed, vortexed for 20 seconds and then subjected tosize analysis using the Malvern Laser Light Scattering instrument. Theresults are shown in Table 1.

TABLE 1 Stock Precursor Solution Ethanol No Passenger Size 1 ml   0 ml[Control] 278 nm 1 ml 0.2 ml [Mixed Population] 242 nm 1 ml 0.5 ml [NLPAssembly] 186 nm 1 ml 0.8 ml [NLP Assembly] 160 nm 1 ml 1.0 ml [NLPAssembly] 138 nm 1 ml 2.0 ml [NLP Assembly] 110 nm 1 ml 3.0 ml [NLPAssembly]  98 nm 1 ml 5.0 ml [NLP Assembly]  61 nm 1 ml  10 ml [NLPAssembly]  34 nm

Example 2 Nanolipidic Particles with Lipid-Soluble Passenger Molecules

Shelf-Stable Precursor Solution (Manufacturing Lot 0013-040010) was usedto prepare NLPs of a variety of defined size populations. Using theMalvern Laser Light Scattering instrument (Zetasizer 1000) nascentpopulations of NLP were measured to be 9 nm. To prepare NLP populationswith Vitamin K1 of defined sizes the following procedure was followed:To 10 ml of Shelf-Stable Stock Solution (Lot 0013-040010) was added 10mg of Vitamin K1. The material was solubilized by vortexing yielding aloaded NLP preparation with an effective concentration of 1 mg/ml ofVitamin K1. 1 ml aliquots of The Vitamin K1 Loaded NLPs were placed in10 ml test tubes. To prepare defined populations of NLP product variousamounts of ethanol were added to each of the sample preparations.Following formation of the NLP preparations, 1 ml aliquots of eachsample were added to 20 ml of distilled water using a 2 ml pipette. Eachpreparation was stirred at room temperature for 5 minutes and then 2 mlaliquots were removed, vortexed for 20 seconds and then subjected tosize analysis using the Malvern Laser Light Scattering instrument. Theresulting Vitamin K1 preparations are shown in Table 2.

TABLE 2 Stock Precursor Solution Ethanol Vitamin K1 Size 1 ml 0 ml[Control] 271 nm 1 ml 0.5 ml [Mixed Population] 229 nm 1 ml 1.0 [NLPAssembly]  76 nm 1 ml 2.0 [NLP Assembly]  51 nm 1 ml 5.0 [NLP Assembly] 40 nm 1 ml 10.0 [NLP Assembly]  30 nm

Example 3 NLPs with Water Soluble Passenger Molecules

Shelf-Stable Precursor Solution (Manufacturing Lot 0013-040010) was usedto prepare NLPs of a variety of defined size populations. Using theMalvern Laser Light Scattering instrument (Zetasizer 1000) nascentpopulations of NLP were measured to be 9 nm. To prepare NLP populationswith Erythromycin of defined sizes the following procedure was followed:A stock solution (150 ml) of Erythromycin 1 mg/ml was prepared byaddition of Erythromycin into 150 ml of distilled water. The solutionwas solubilized by stirring at room temperature for 5 minutes. 1 mlaliquots of NLP starting materials were placed in 10 ml test tubes. Toprepare defined populations of NLP product incorporating Erythromycinvarious amounts of ethanol were added to each of the samplepreparations. Following formation of the NLP preparations, 1 ml aliquotsof each sample were added to 20 ml of the prepared Erythromycin stocksolution in water 1 mg/ml using a 2 ml pipette. Each preparation wasstirred at room temperature for 5 minutes and then 2 ml aliquots wereremoved, vortexed for 20 seconds and then subjected to size analysisusing the Malvern Laser Light Scattering instrument. Results are shownin Table 3.

TABLE 3 Stock Precursor Solution Ethanol Erythromycin Size 1 ml   0 ml[Control] 289 nm 1 ml  0.5 ml [NLP Assembly] 162 nm 1 ml  1.0 ml [NLPAssembly] 141 nm 1 ml  2.0 ml [NLP Assembly] 105 nm 1 ml  5.0 ml [NLPAssembly]  66 nm 1 ml 10.0 ml [NLP Assembly]  52 nm

Example 4 NLPs with Both Lipid and Water Soluble Passenger Molecules

Shelf-Stable Precursor Solution (Manufacturing Lot 0013-040010) was usedto prepare NLPs of a variety of defined size populations. Using theMalvern Laser Light Scattering instrument nascent populations of NLPwere measured to be 9 nm. To prepare NLP populations with both VitaminB12 (water soluble) and Vitamin K1 (lipid soluble) of defined sizes thefollowing procedure was followed: A stock solution (70 ml) of VitaminB12 1 mg/ml was prepared by addition of 70 mg of Vitamin B12 into 70 mlof distilled water. The solution was solubilized by stirring at roomtemperature for 5 minutes. A preparation of Vitamin K1 loaded NLPs wereprepared by solubilization of 10 mg of Vitamin K1 into 10 ml ofShelf-Stable Precursor Solution Manufacturing Lot 0013-040010). 1 mlaliquots of NLP loaded with Vitamin K1 were placed in 10 ml test tubes.To prepare defined populations of NLP product incorporating both VitaminK1 and Vitamin B12 various amounts of ethanol were added to each of thesample preparations. Following formation of the NLP preparations, 0.5 mlaliquots of each sample were added to 10 ml of the prepared Vitamin B12stock solution in water 1 mg/ml using a 2 ml pipette. Each preparationwas stirred at room temperature for 5 minutes and then 2 ml aliquotswere removed, vortexed for 20 seconds and then subjected to sizeanalysis using the Malvern Laser Light Scattering instrument. Theresults obtained are shown in Table 4.

TABLE 4 Stock Precursor Solution Ethanol Vitamins K1 and B12 Size 1 ml 0ml [Control] 217 nm  1 ml 0.5 [NLP Assembly] 122 nm  1 ml 1.0 [NLPAssembly] 94 nm 1 ml 2.0 [NLP Assembly] 71 nm 1 ml 5.0 [NLP Assembly] 68nm 1 ml 10.0 [NLP Assembly] 57 nm

Example 5 Preparation of Defined Populations—Effect of Solvent

Shelf-Stable Precursor Solution (Lot number 0013-040100) was used asstarting stock for production of NLP assemblies

Various amounts and types of solvent were added to the starting stock tomake solvent diluted stock solutions. NLP assemblies were prepared byconverting the solvent diluted stock solutions by addition of 1 mlthereof into 20 ml of distilled water while stirring, using a mixingplate and stir bar at room temperature. The mixture was stirred for 5minutes, vortexed gently for 20 seconds, and placed into a sample cuvetwhich was used to determine the size of the resulting NLP assemblypopulation with a Malvern 1000 Zetasizer™ laser light scatteringinstrument set to evaluate particle size using multimodal analysis.

In addition the size of the native NLPs present in the precursor werealso determined by examining the precursor alone using the Malvern 1000with settings as already described.

The data in Table 5 demonstrates that various solvents may be used toform NLP assemblies or NLPs. While Ethanol will likely be chosen formany applications due to it relatively non-toxic properties, it may bedesirable to use other solvents for particular NLP characteristics.

TABLE 5 Water Solvent Amount Stock Amount B Added Precursor Added to 1ml Ethanol Methanol 1-Propanol 2-Propanol Solution A to A of A + BSolvent Solvent Solvent Solvent (mls) (mls) (mls) Size (nm) Size (nm)Size (nm) Size (nm) 1 0 0 14 14 14 14 1 0 20 240 240 240 240 1 0.5 20133 191 112 95 1 1 20 ND ND ND 46 1 2 20 108 97 66 13 1 5 20 49 60 23 ND1 10 20 25 43 ND ND

Example 6 Use of Heating to Control Size

NLP assemblies and ECVs which have been prepared by secondary additionof solvent to precursor solution such as in Example 5 may be heated tofurther reduce (in a controlled manner) the average size thereof.

NLP assemblies and ECVs were produced using Stock Precursor Solution(Lot number 0013-040010) which contained native NLPs sized atapproximately 10 nanometers (as measured using the Malvern 1000Zetasizer laser Light Scattering Instrument with sizing for particlepopulations set to multimodal setting).

Dilutions with ethanol were made of the control and test portions asindicated in Table 6 to form solvent diluted stock solutions. NLPassemblies and ECVs were prepared by converting the solvent dilutedstock solutions by addition of 1 ml thereof to 20 ml of distilled waterwhile stirring, using a mixing plate and stir bar at room temperature asdescribed in Example 5. One portion was retained as a control, and oneportion was removed for testing of the determination of the effect ofheating on the NLP assembly population or ECV population.

Heating of the test portion was accomplished by placing five ml samplesinto a test tube which was then lowered into rapidly boiling water for 3minutes. The tubes containing the thus-heated samples were removed fromboiling water and allowed to cool to room temperature, then vortexedgently for 20 seconds. The control was treated in the same way with theexception that it was not heated.

An aliquot of each portion (control and heated) was transferred into asample cuvet which was used to determine the size of the resulting NLPassembly population with a Malvern 1000 Zetasizer™ laser lightscattering instrument set to evaluate particle size using multimodalanalysis.

TABLE 6 Water Stock (Added Size Precursor Ethanol to 1 ml of (nm)Solution A B A + B) No Size (nm) % Reduction (mls) (mls) (mls) HeatingWith Heating with Heating 1 0 0 10 10 0 1 0 20 252 158 37.3 1 0.5 20 188125 33.5 1 2 20 138 96 30.4 1 5 20 57 35 38.6 1 10 20 36 26 31

The results of these evaluations demonstrate that heating of NLPassemblies or ECVs reduced the size of resulting carrier vehicles in acontrolled manner with an average reduction of 34%. Because thesewithstand heat, it is possible to not only reduce the size of the NLPsbut to use heating and sterilizing techniques such as flashpasteurization, which is desirable in the food and beverage industry tocontrol the bacterial load. This can be conducted at temperatures up toabout 220 degrees Fahrenheit with the usual range of about 180 to 220degrees Fahrenheit.

Example 8 Masking the Taste of Lidocaine Hydrochloride

Encapsulating particles were prepared using Shelf-Stable Precursor Stock(Lot Number 0013-050015). The size of native NLPs in this precursorstock was determined to be 8.1 nm using the Malvern 1000 Zetasizer LaserLight Scattering Instrument set to analyze size populations usingmultimodal analysis mode. To 5 ml of stock was added 50 mg of LidocaineHydrochloride. The mixture was vortexed until the LidocaineHydrochloride was dissolved into the stock, forming preloaded NLPs.Ethanol was added to the preloaded NLPs as indicated below to formsolvent diluted NLP stock. One ml of solvent diluted NLP stock wasfurther diluted by addition to 20 ml of distilled water while stirringusing mixing plate and stir bar at room temperature. The resultingcarrier vehicles were stirred for 5 minutes and vortexed gently for 20seconds and placed into a sample cuvet which was used to determine thesize of the resulting population with a Malvern 1000 Zetasizer™ laserlight scattering instrument set to evaluate particle size usingmultimodal analysis.

TABLE 8 Water (Added to 1 ml of Preloaded with NLP stock A Ethanol B A +B) Lidocaine (mls) mls) (mls) Hydrochloride? Size (nm) 1 0 0 No 8.1 1 020 No 262 1 0 20 Yes 276 1 0.5 20 Yes 91 1 1 20 Yes 88 1 2 20 Yes 68 1 520 Yes 46

The size of the native NLPs present in the stock precursor wasdetermined by examining the precursor alone using the Malvern 1000 withsettings as already described.

The preparations of carrier vehicles formed using the Preloaded NLPswith Lidocaine Hydrochloride masked the taste of the LidocaineHydrochloride.

Example 9 Caffeine as a Passenger Molecule

Carrier vehicle preparations were made using caffeine as the passengermolecule. Two concentrations were evaluated for the ability to masktaste and for stability over time both in size and in taste maskingcapability.

A Preloaded NLPs stock preparation was prepared. First, 5 g of caffeinewas dissolved into 100 ml of distilled water. Separately, 10 ml of Shelfstable precursor solution (Lot number 0013-010007), which containednative NLPs measured at 12 nm using the Malvern 1000 Zetasizer LaserLight Scattering Instrument with analysis mode set to multimodalpopulation setting, was diluted with 10 ml of Ethanol as a secondarysolvent (1:1 Volume/Volume dilution). This formed solvent diluted NLPstock. The solvent diluted NLP stock was added to the 100 ml solutioncontaining 5 grams of caffeine while the solution was being stirred atroom temperature. The entire 120 ml preparation thus prepared was addedinto 880 ml of distilled water while stirring at room temperature for 5minutes. A 3 ml sample was removed for analysis and vortexed gently for20 seconds. The carrier vehicles loaded with caffeine as a passengermolecule were placed into a sample cuvet which was used to determine thesize of the resulting carrier vehicle population with a Malvern 1000Zetasizer™ laser light scattering instrument set to evaluate particlesize using multimodal analysis. The carrier vehicles were determined tobe 121 nm.

When the sample was evaluated for taste masking, the bitter taste of thecaffeine was masked. In addition, the final preparation of carriervehicles loaded with caffeine was optically clear.

It was concluded that this procedure could be used for applicationscalling for optically clear products suitable for oral consumption suchas beverages or medicinal preparations. Bitter or otherwiseobjectionable passenger molecules thus encapsulated in carrier vehiclescould be consumed without the consumer having to taste the passengermolecule. In addition to caffeine, it is contemplated that electrolytes(NaCl and KCl) and vitamins among other passenger molecules would beappropriate candidates for formulation as described in Example 8.

Example 10 Production of Preloaded NLP Preparations Containing Caffeine

Preloaded NLPs were prepared from Shelf-Stable Precursor stock (LotNumber 0013-050015). The size of the nascent NLPs was determined to be 8nm using the Malvern 1000 Zetasizer Laser Light Scattering Instrumentset to analyze size populations using multimodal analysis mode. Thepreparation containing caffeine was prepared as follows:

1. Anhydrous Caffeine (2.5 g) was dissolved into 50 ml of distilledwater at room temperature

2. To 20 ml of Precursor Stock was added 10 ml of ethanol.

3. Material from step 2 was added to caffeine solution from step 1 whilestirring at room temperature. The mixture was stirred for 5 minutes.This resulted in the production of preloaded NLPs containing caffeine at31.3 mg/ml.

4. The finished product containing caffeine at 0.5 mg/ml was produced bymixing preparation from step 3 into an appropriate volume of distilledwater (1:62.5 volume/volume)

5. The size of the finished preparation was determined using the Malvern1000 Zetasizer Laser Light Scattering Instrument set to analyzepopulations using multimodal analysis mode. The size of the finishedpreparation was determined to be 147 nm. The finished preparationcontaining 0.5 mg/ml caffeine was optically clear.

6. The finished preparation from step 5 was evaluated for its ability tomask the taste of caffeine by placing 1 ml of preparation into mouth.The preparation was found to effectively mask the taste of caffeine.

Example 11 Production of Preloaded NLP Preparations Containing LipidSoluble Vitamins as a Means to Produce Finished Products of Small Sizeand Ability to Mask Adverse Taste of Lipid Soluble Vitamins

Preloaded NLPs were prepared from Shelf-Stable Precursor Stock (LotNumber 0013-050015). The size of the nascent NLPs was determined to be 8nm using the Malvern 1000 Zetasizer Laser Light Scattering Instrumentset to analyze size populations using multimodal analysis mode. Thepreparation containing lipid soluble vitamins was prepared as follows:

1. Solvent diluted precursor stock was prepared by adding 20 ml ofethanol to 40 ml of shelf stable precursor stock.

2. 250 mg of Vitamin D3 DSM Chemicals), 500 mg of Vitamin E (DSMChemicals) and 4 mg of Vitamin K (DSM Chemicals) were dissolved in thepreparation resulting from step one. This was stirred at roomtemperature and resulted in a preloaded NLP population.

3. The preloaded NLP preparation from step 2 was diluted 1 to 50 intodistilled water to yield a finished product suitable for oralconsumption containing the recommended daily allowance (“RDA”) of the 3lipid soluble vitamins.

4. The size of the finished preparation was determined using the Malvern1000 Zetasizer Laser Light Scattering Instrument set to analyzepopulations using multimodal analysis mode. The size of the finishedpreparation was determined to be 168 nm.

5. The finished preparation from step 5 was evaluated for its ability tomask the taste of offensive taste of the three lipid soluble vitamins byplacing 1 ml into the mouth. The preparation was found to effectivelymask the taste of all three lipid soluble vitamins; Vitamin D3, E and K.

I claim:
 1. NLPs (Nanolipidic Particles) comprising particles formedfrom a hydrous monophasic precursor solution having phospholipids,water, and a non-aqueous solvent selected from the group consisting ofethanol, 1-propanol, and 2-propanol, said hydrous monophasic precursorsolution having been diluted by one or more sequential dilutions ofnon-aqueous solvent resulting in a solvent-diluted precursor solution,and then dilution of said solvent-diluted precursor solution with anaqueous solvent to yield said NLPs sized from about 1 nm to about 20 nm,said NLPs being self-stabilizing at this size range by the retention ofsaid aqueous solvent in said NLPs, said NLPs further retaining aconcentration of said non-aqueous solvent from about 0.5% to about 14%.2. The NLPs of claim 1, wherein the NLPs are sized from about 6 to about12 nm.
 3. The NLPs of claim 1, wherein said non-aqueous solvent isethanol.
 4. The NLPs of claim 1, wherein said phospholipids are selectedfrom the group consisting of phosphatidyl choline (PC),phospatidylethanolamine (PE), phosphatidic acid (PA) andphosphatidylinositol (PI), and mixtures thereof.
 5. The NLPs of claim 1,wherein said non-aqueous solvent is ethanol, and said phospholipids areselected from the group consisting of phosphatidyl choline (PC),phospatidylethanolamine (PE), phosphatidic acid (PA) andphosphatidylinositol (PI), and mixtures thereof.
 6. The NLPs of claim 5,wherein the NLPs are sized from about 6 to about 12 nm.
 7. The NLPs ofclaim 1, wherein said NLPs are for oral consumption.
 8. The NLPs ofclaim 5, wherein said NLPs are for oral consumption.
 9. The NLPs ofclaim 1, wherein said NLPs are for topical application.
 10. The NLPs ofclaim 5, wherein said NLPs are for topical application.
 11. NanolipidicParticles (NLPs) having a passenger molecule, said NLPs comprisingparticles formed from a hydrous monophasic precursor solution havingphospholipids, water, and a non-aqueous solvent selected from the groupconsisting of ethanol, 1-propanol, and 2-propanol, said hydrousmonophasic precursor solution having been diluted by one or moresequential dilutions of non-aqueous solvent resulting in asolvent-diluted precursor solution having said NLPs sized from about 1nm to about 20 nm, and then said solvent diluted-precursor solutionbeing diluted with an aqueous solvent, said NLPs being stabilized for atleast two years at this size range by the retention of said aqueoussolvent in said NLPs, said NLPs further retaining a concentration ofsaid non-aqueous solvent from about 0.5% to about 14%, with a passengermolecule encapsulated therein.
 12. The NLPs of claim 11, wherein saidnon-aqueous solvent is ethanol.
 13. The NLPs of claim 11, wherein saidphospholipids are selected from the group consisting of phosphatidylcholine (PC), phospatidylethanolamine (PE), phosphatidic acid (PA) andphosphatidylinositol (PI), and mixtures thereof.
 14. The NLPs of claim11, wherein the NLPs are sized from about 6 to about 12 nm.
 15. The NLPsof claim 11, wherein said NLPs are for oral consumption.
 16. The NLPs ofclaim 11, wherein said NLPs are for topical application.
 17. The NLPs ofclaim 11, wherein said non-aqueous solvent is ethanol, and saidphospholipids are selected from the group consisting of phosphatidylcholine (PC), phospatidylethanolamine (PE), phosphatidic acid (PA) andphosphatidylinositol (PI), and mixtures thereof.
 18. The NLPs of claim17, wherein said NLPs are for oral consumption.
 19. The NLPs of claim17, wherein said NLPs are for topical application.
 20. NanolipidicParticles (NLPs), said NLPs comprising particles formed from a hydrousmonophasic precursor solution having phospholipids, water, and anon-aqueous solvent selected from the group consisting of ethanol,1-propanol, and 2-propanol, said hydrous monophasic precursor solutionhaving been diluted by one or more sequential dilutions of non-aqueoussolvent and then at least one dilution of aqueous solvent yielding saidNLPs sized from about 6 nm to about 12 nm, said NLPs having a retentionof said aqueous solvent in said NLPs, said NLPs further retaining aconcentration of said non-aqueous solvent from about 0.5% to about 14%.21. The NLPs of claim 20, wherein said non-aqueous solvent is ethanol.22. The NLPs of claim 20, wherein said phospholipids are selected fromthe group consisting of phosphatidyl choline (PC),phospatidylethanolamine (PE), phosphatidic acid (PA) andphosphatidylinositol (PI), and mixtures thereof.
 23. The NLPs of claim20, wherein said NLPs are for oral consumption.
 24. The NLPs of claim20, wherein said NLPs are for topical application.
 25. The NLPs of claim20, wherein said non-aqueous solvent is ethanol, and said phospholipidsare selected from the group consisting of phosphatidyl choline (PC),phospatidylethanolamine (PE), phosphatidic acid (PA) andphosphatidylinositol (PI), and mixture thereof.
 26. The NLPs of claim25, wherein said NLPs are for oral consumption.
 27. The NLPs of claim25, wherein said NLPs are for topical application.
 28. NLPs (NanolipidicParticles) comprising particles formed from a hydrous monophasicprecursor solution having phospholipids, water, and a non-aqueoussolvent selected from the group consisting of ethanol, 1-propanol, and2-propanol, said hydrous monophasic precursor solution having beendiluted by one or more sequential dilutions of non-aqueous solvent, andthen at least one dilution with aqueous solvent, said dilutions yieldingNLPs stably sized from 1 nm to 20 nm, said NLPs retaining said aqueoussolvent in said NLPs ranging from a 1:1 to 20:1 ratio of water tophospholipids, said NLPs further retaining having a concentration ofsaid non-aqueous solvent from about 0.5% to about 14%.
 29. The NLPs ofclaim 28, wherein the NLPs are sized from 6 nm to 12 nm.
 30. The NLPs ofclaim 28, wherein said non-aqueous solvent is ethanol.
 31. The NLPs ofclaim 28, wherein said phospholipids are selected from the groupconsisting of phosphatidyl choline (PC), phospatidylethanolamine (PE),phosphatidic acid (PA) and phosphatidylinositol (PI), and mixturethereof.
 32. The NLPs of claim 28, wherein said non-aqueous solvent isethanol, and said phospholipids are selected from the group consistingof phosphatidyl choline (PC), phospatidylethanolamine (PE), phosphatidicacid (PA) and phosphatidylinositol (PI), and mixtures thereof.
 33. TheNLPs of claim 28, wherein a passenger molecule is encapsulated withinsaid NLPs.
 34. NLPs (Nanolipidic Particles) comprising particles formedby a plurality of sequential dilutions of a hydrous monophasic precursorsolution, said hydrous monophasic precursor consisting essentially ofphospholipids, water and a non-aqueous solvent selected from the groupcontaining ethanol, 1-propanol and 2-propanol, and said sequentialdilution including one or more dilutions with a non-aqueous solvent andthen at least one dilution with an aqueous solvent, yielding NLPs stablysized from 1 nm to 20 nm, said NLPs self-stabilizing at this size rangeby the incorporation and retention of said aqueous solvent in said NLPs,said NLPs further retaining a concentration of non-aqueous solvent fromabout 0.5% to about 14%, and said NLPs are independently shelf-stable atleast two years.
 35. The NLPs of claim 34, wherein the NLPs are sizedfrom 6 nm to 12 nm.
 36. The NLPs of claim 34, wherein said non-aqueoussolvent is ethanol.
 37. The NLPs of claim 33, wherein said phospholipidsare selected from the group consisting of phosphatidyl choline (PC),phospatidylethanolamine (PE), phosphatidic acid (PA) andphosphatidylinositol (PI), and mixture thereof.
 38. The NLPs of claim34, wherein said non-aqueous solvent is ethanol, and said phospholipidsare selected from the group consisting of phosphatidyl choline (PC),phospatidylethanolamine (PE), phosphatidic acid (PA) andphosphatidylinositol (PI), and mixtures thereof.
 39. The NLPs of claim34, wherein a passenger molecule is encapsulated within said NLPs.